Lactic acid bacteria with anxiolytic properties and uses thereof

ABSTRACT

The invention concerns lactic acid bacteria with anxiolytic properties and not inducing sedative effect. Said lactic acid bacteria are useful in particular for preparing foods or medicines.

The invention relates to novel strains of lactic ferments and to theiruse for the production of foods endowed with anxiolitic properties.

Stress, which is a defensive reaction of the body against externalattacks, causes in particular at the level of the nervous system,biological disruptions which can develop into various pathologicalconditions. The latter can manifest themselves directly at thepsychological level, in particular in the form of anxiety, and/or cantake the form of somatic manifestations, such as high blood pressure,gastric ulcerations and the like. They frequently progress to chronicstates.

To treat the consequences of stress, whether involving anxiety orsomatic manifestations, anxiolytic, and in particular benzodiazepines,are frequently used. However, these medicaments, which can rapidly causea phenomenon of addiction, may not be suitable for a long-termtreatment. Moreover, their anxiolytic action is accompanied inparticular by sedative properties which can cause unwanted side effects.

Some research studies have demonstrated the existence, in milk or inproducts derived therefrom, of substances endowed with pharmacologicalproperties toward pathological conditions linked to stress. Inparticular, PCT application WO 98/05343, in the names of CALPIS CO. ANDGROUPE DANONE, describes an agent possessing antistress propertiesmanifested by an antihypertensive effect. This agent is obtained byfermenting milk with a lactic acid bacterium of the genus Lactobacillus,in particular Lactobacillus helveticus. It is resistant to sterilizationand can be used to prepare foods participating in the prevention and/ortreatment of the peripheral (somatic) effects of stress, and which canbe consumed in the context of the usual diet, regularly and over longperiods. No central, in particular anxiolytic, effect resulting from theadministration of this agent is demonstrated in PCT application WO98/05343.

Application EP 95402697 in the name of: SOCIETE COOPERATIVE LAITIEREAGRICOLE D'ARTOIS ET DES FLANDRES, LA PROSPERITE FERMIERE, describes adecapeptide resulting from the tryptic hydrolysis of casein csl. Theparental administration of this decapeptide, or of the tryptichydrolysate comprising it induces an anxiolytic and anticonvulsanteffect of the same type as that by the benzodiazepines. On the otherhand, this document provides no information relating to the activity ofthis hydrolysate when administered orally, and the possibility ofincorporating it into foods in order to confer antistress properties onthem.

The inventors have now demonstrated, in dairy products fermented bycertain strains of lactic acid bacteria, the existence of one or moreactive ingredients capable of inducing, when they are administeredorally, an anxiolytic-type antistress effect, without possessing thesedative properties of the benzodiazepines.

The subject of the present invention is also the use of at least one ofthe newly isolated and characterized strains of lactic acid bacteriamentioned above, for the production of a nonsedative anxiolytic.

The subject of the present invention is a strain of lactic acidbacterium chosen from the group consisting of:

the Streptococcus thermophilus S242 strain deposited on Feb. 24, 1999 inaccordance with the Budapest Treaty at the CNCM (Collection Nationale deCultures de Micro-organismes), 28 rue du Docteur Roux, 75724 Paris,under the number I-2130;

the Streptococcus thermophilus S003 strain deposited on 24 February 1999in accordance with the Budapest Treaty at the CNCM (Collection Nationalede Cultures de Micro-organismes), 28 rue du Docteur Roux, 75724 Paris,under the number I-2129;

the Lactobacillus gasseri L012 strain deposited on Feb. 24, 1999 in,accordance with the Budapest Treaty at the. :-CNCM (Collection Nationalede Cultures de Micro-organismes), 28 rue du Docteur Roux, 75724 Paris,under the number I-2131;

the Lactobacillus acidophilus L030 strain deposited on Feb. 24, 1999 inaccordance with the Budapest Treaty at the CNCM (Collection Nationale deCultures de Micro-organismes), 28 rue du Docteur Roux, 75724 Paris,under the number I-2132.

The subject of the present invention is also the newly isolated andcharacterized strains of lactic acid bacteria mentioned above, which canbe used for the production of a nonsedative anxiolytic.

The characteristics of the strains of lactic acid bacteria in accordancewith the invention are summarized in tables I and II below.

TABLE I Strain S003 S242 L012 L030 CNCM No. I-2129 I-2130 I-2131 I-2132Morphology Fairly large Small cocci Small fine Medium- cocci in pairs orbacilli, sized predominantly in the form faintly bacilli, in the form ofshort colored, isolated of long chains often coco- or in the chains anda bacillary form of few in the short form of chains medium and shortchains Gram + Gram + Gram + Gram + Metabolism Oxidase:- Oxidase:-Oxidase:- Oxidase:- Catalase:- Catalase:- Catalase:- Catalase:-Facultative Facultative Nitrate Nitrate aero- aero- reductase:-reductase:- anaerobe anaerobe Nitrite Nitrite reductase:- reductase:-Aero- Aero- anaerobe anaerobe Growth T ° 37-44° C. 37-44° C. 37-44° C.37-44° C.

TABLE II Strain S003 S242 L012 L030 CNCM No. I-2129 I-2130 I-2131 I-2132Galactose − − + + D-Glucose + + + + D-Fucose − − + + D-Mannose − − + +N-Acetyl-Glucosamine − − + + Amygdalin − − + + Arbutin − − +/− + Esculin− − + + Salicin − − + + Cellobiose − − + + Matose − − + +Sucrose + + + + Threalose − − + + β-Gentobiose − − + + D-Tagatose − − +− D-Turanose − − − + Lactose + + + + D-Raffinose − − − + Melibiose − −− + α-Methyl-D-Glucoside − − − +

The present invention relates in particular to the use, for theproduction of a nonsedative anxiolytic, of at least one Streptococcusthermophilus strain chosen from the S242 strain and the S003 strain,combined with the S. thermophilus S147 strain deposited at the CNCM onDec. 30, 1994 under the number I-1520.

The present invention also relates to lactic ferments comprising atleast one strain in accordance with the invention, advantageouslycombined with another strain of lactic acid bacterium.

According to a preferred embodiment of a lactic ferment in accordancewith the invention, it comprises at least the S. thermophilus S242strain and/or the S. thermophilus S003 strain, advantageously combinedwith the S. thermophilus S147 strain.

The subject of the present invention is also a non-sedative anxiolyticcomprising at least one strain of lactic acid bacterium in accordancewith the invention as defined above, or capable of being obtained from aculture of said strain.

For the production of a nonsedative anxiolytic in accordance with theinvention, at least one strain of lactic acid bacterium in accordancewith the invention is cultured; in particular a lactic fermentcomprising at least the S. thermophilus S424 strain and/or the S.thermophilus S003 strain advantageously combined with the S.thermophilus S147 strain, and optionally with the L. gasseri L012 strainand/or the L. acidophilus L030 strain, is cultured in an appropriatemedium, that is to say a medium comprising at least one substrateallowing the growth of said bacteria. Said culture medium is preferablymilk or a milk-based medium. It may be chosen in particular from themilks of the various species of mammals, optionally semiskimmed orskimmed, the products resulting from the dilution or the concentrationof these milks, such as for example ultrafiltration of diafiltrationretentates, milk-based media, such as for example bases for milk-basedfoods, bases for yogurt and the like. These milks may, in addition, besupplemented with, for example, lactose, minerals, vitamins, fattysubstances or otherwise, hydrophilic milk solids, soybean proteins,plant extracts and the like.

This medium is inoculated with a culture comprising at least one strainof lactic acid bacterium in accordance with the invention, preferablycomprising 10⁷ to ₁₀ ⁹ bacteria/ml. The optimum culture conditions varyaccording to the strain or the mixture of strains involved. For example,if the culture for inoculation consists of a strain or a mixture ofstrains of Streptococcus thermophilus, the culture will be carried outfor about 24 h and the optimum culture temperature will be between 30and 44° C.; if the culture for inoculation consists of a strain or amixture of strains of L. acidophilus or of L. gasseri, the culture willbe carried out for about 16 h and the optimum culture temperature willbe between 37 and 44° C. In the case where the culture for inoculationcombines at least one Streptococcus thermophilus strain with at leastone L. acidophilus or L. gasseri strain, the culture will be carried outfor 16 to 24 h, and the optimum culture temperature will be between 37and 44° C.

The bacterial culture thus obtained possesses the properties of anonsedative anxiolytic in accordance with the invention.

The nonsedative anxiolytics in accordance with the invention are activewhen administered orally. Regular administration thereof causes, after afew days, a reduction in anxiety, with no reduction in activity; it canbe extended for a very long period, with no addiction effect, andwithout the appearance of unfavorable side effects. These properties, aswell as the fact that it is possible to obtain them from micro-organismsbelonging to species commonly used in the food industry, and from rawmaterials which are also intended for dietary use, make it possible touse them in the context of the manufacture of foods or dietarysupplements, whose consumption exerts a beneficial effect in the contextof the treatment or of the prevention of stress-related pathologicalstates.

The subject of the present invention is also the food products ordietary supplements comprising a non-sedative anxiolytic in accordancewith the invention.

These food products may be in particular products comprising at leastone strain of lactic acid bacterium or a lactic ferment in accordancewith the invention. They are in particular products which can beobtained by fermenting milk or a milk-based medium with said strain ofsaid ferment. Food products in accordance with the invention can also beprepared by adding to any food which is fermented or otherwise apreparation comprising a nonsedative anxiolytic in accordance with theinvention.

The beneficial effects of the foods in accordance with the inventiongenerally appear starting from a dose of 5 ml/day/kg of weight, in thecase of a fermented milk containing a total population (which mayconsist of one or more strains of bacteria in accordance with theinvention) of lactic acid bacteria in accordance with the invention ofat least 10⁵ CFU/ml. Substantially higher doses can however be taken,with no harmful side effects.

The present invention will be understood more clearly with the aid ofthe additional description which follows, which refers to nonlimitingexamples illustrating the anxiolytic properties of fermented dairyproducts obtained in accordance with the invention.

EXAMPLE 1 Preparation of Various Test Dairy Products

The strains and ferments used in the various experiments which followare the following:

S. therimophilus S242 strain

S. thermophilus S147 strain

S. thermophilus S003 strain

Ferment F042 (ferment combining the 3 strains

S242, S147, S003 of S. thermophilus)

L. gasseri L012 strain

L. acidophilus L030 strain.

Manufacture of the Products:

UHT milk containing 0% fat was treated at 95° C. for 10 minutes, andthen cooled to 37 or 44° C. depending on the temperature for thefermentations, before being inoculated with active cultures of thestrains used to obtain the desired preparation.

The inoculation with the S. thermophilus S242, S147 and S003 strainsused individually, is carried out at the rate of 1% (v/v) of a culturecontaining 10⁸ to 10⁹ CFU/ml of sterile milk supplemented with yeastautolysate (3 h at 44° C.).

The inoculation with the ferment S042 is carried out at the rate of 30g/100 1 of a frozen concentrated mixture between 10⁸ and 10⁹ CFU/ml ofthe S242, S147 and S003 strains. The incubation is carried out at 44°C.; when the pH reaches about 4.5, the preparation is transferred to 4°C. and stored at this temperature pending use.

The bacterial populations at the end of the fermentation in the productsfermented by the strains separated are: Streptococcus thermophilus S242:1.2×10⁹ CFU/ml; Streptococcus thermophilus S147: 1.8×10⁹ CFU/ml;Streptococcus thermophilus S003: 1.3×10⁷ CFU/ml.

In the product fermented by the F042 mixture, the Streptococcusthermophilus (S242+S147+S003) concentration is 1.9×10⁸ CFU/ml.

The inoculation with the L. acidophilus L012 and L030 strains is carriedout at the rate of 3% (v/v) of a culture containing 10⁸ to 10⁹ CFU/ml inneutral MRS medium (16 h at 37° C.). The incubation is carried out at37° C.; when the pH reaches a value of between 4.5 and 5.9, thepreparation is transferred to 40° C., and stored at this temperaturepending use.

The filtered whey of the milk fermented by the ferment F042 is preparedby centrifuging 500 ml of fermented milk at 10 000 rpm (Sorval RC5Bcentrifuge, GSA rotor) twice 15 minutes and then by filtering thesupernatant over a NALGENE filter made of cellulose nitrate(HIGH-BINDING PROTEIN) 0.45 μm and then 0.22 μm.

EXAMPLE 2 Evaluation of the Anxiolytic Effect of a Mixture of 3Thermophilic Streptococci

The Products Tested are the Following:

3S: milk fermented by the ferment F042 (comprising 3 S. thermophilusS242, S147, S003)

3S+P: milk fermented by the ferment F042+Passionflower

3ST: milk fermented by the ferment F042 and then thermized at 73° C. for30 seconds with homogenization at 100 bar.

L: control milk sterilized at 130° C. for 30 seconds.

The anxiolytic effect is evaluated using the Conditioned DefensiveBurying model, which was developed from the procedure by J. P. PINEL andD. TREIT (PINEL and TREIT, J. Comp. & Physiol. Psychol., 92, 708-712,1978; TREIT et al., Pharmacol. Biochem. Behav., 15, 619-626, 1981). Thismodel uses the natural propensity of most rodents to bury themselvesfrom a stressful source of stimulus (TREIT, Pharmacol. Biochem. Behav.,22, 1, 47-52, 1985; MEERT and COLPAERT, Psychopharmacology, 88, 445-450,1986; CRAFT et al., Pharmacol. Biochem. Behav., 30, 3, 775-780, 1988;TREIT, Pharmacol. Biochem. Behav., 36, 203-205, 1990).

Animals

Seventy two SPF Wistar/AF male rats (IFFA-CREDO breeding center,69—St-Germain sur l'Arbresle, France) weighing 320 to 340 g were used.On reception, the rats were weighed, marked and divided into groups of 4in type F polycarbonate cages (48×27×20 cm, U.A.R., 91—Epinay-Sur-Orge,France). The animals were stalled in an air-conditioned animal house, ata temperature of 22-24° C. Food (M25 biscuits, Ets PIETREMENT,77—Provins, France) and drink were available to the rats ad libitum.They were subjected to a light-darkness cycle of 12 hours (light from 24h to 12 h).

After a week's familiarization with the laboratory conditions, the 72rats were randomly divided into 6 groups (n=12). The rats in thedifferent groups were all handled in the same way and under the sameconditions.

Materials

Experimental Cage

Habituation and the tests were carried out in a transparent cage havingthe dimensions 44×28×18 cm, whose base is covered with sawdust over athickness of 5 cm. In the middle of a lateral face, an orifice allowsthe attachment of an electric probe, 2 cm above the level of thesawdust.

Electric Probe

It is a probe made of plexiglas having the dimensions 7×2×0.5 cm,covered with a copper printed circuit in which the gap between the wiresis 1 mm, chosen so that one leg of the rat coming to rest on the probeeasily makes contact between them, thus allowing the passage of current.

Electrical Apparatus

The probe is connected to a manually triggered generator of electricshocks of 0 to 8 mA (OPEN-SYSTEMS, 54—Maxeville, France).

Recording and Evaluation of the Behavioral Sequences

In the faintly illuminated experimental room, a video camera was placedin front of the cage of the setup, at a distance of one meter. Thecamera is connected to a video recorder and to a control monitor, whichare placed in an adjacent room with the electric shock generator.

Experimental Procedure

The experiment was carried out according to a double blind design.

Habituation

During the 2 days preceding the first test, the rats were transportedfrom the animal house to the experimental room and introduced into theexperimental device in order to habituate them to both the handling andto the cage of the device in the absence of the electrode. Each group of4 rats was placed in the device for 20 minutes. The sawdust is changed,leveled to a uniform height of 5 cm, before the period of habituation ofeach of the groups.

Experience

The Conditioned Defensive Burying test is carried out in the first 5hours of the dark phase, a phase during which the rats are the mostactive.

The electric probe is inserted into the cage before the beginning of thefirst test. Each rat is placed in the experimental cage on the sideopposite the probe.

During the first test (test 1), a single electric shock, of lowintensity (2 mA) is delivered to the animal when it places a hind legfor the first time on the probe.

Following the electric shock, the behavior of each rat is recorded for 5minutes. During the next two tests (tests 2 and 3), the rat is againplaced in the experimental cage in the presence of the inactive probe.The probe is cleaned after each rat and the sawdust is changed andleveled to a uniform height of 5 cm before the test session for eachcage of rats.

Administration of the Products

The products L, 3ST, 3S and 3S+P were orally administered daily at thesame times, at the doses of 5 ml/kg by means of syringes provided withintragastric forced-feeding probes (HARVARD APPARATUS, 91—Les Ulis,France). The burying tests (test 1 on day 6 and test 2 on day 8) werecarried out 6 hours after the administration of the products.

The positive control is diazepam (3 mg/kg), which was suspended in anaqueous solution of methyl cellulose at 1% and was orally administereddaily, for the same period. On the test days, it was administered 1 hourbefore the burying test.

The four rats of each cage each received a different product.

Variables

duration of burying for the probe: the burying sequences are remarkablystereotyped after the shock; the rat faces the electric probe and sendsthe sawdust in its direction by a rapid alternating movement of the hindlegs;

number of writhing movements in relation to the probe: the rat stops afew centimeters from the probe and extends its neck in order to bringthe muzzle closer to the probe;

number of approaches to the probe: every approach of the rat's head inthe direction of the probe to a distance of less than 3 cm;

number of flights in relation to the probe: every hasty flight of therat following an approach to or a contact with the probe;

percentage of approaches to the probe followed by flights=(number offlights/number of approaches)×100.

Statistical Analysis

The data were classified in increasing order for the followingvariables: duration of burying, number, of writhing movements andpercentage of approaches followed by flights. Within the variousvariables, a rank was attributed to each data and the rank sumconstitutes the overall stress score for each rat.

The KRUSKAL-WALLIS test was used to demonstrate the existence ofheterogeneity among the groups. The MANN-WHITNEY test served to comparethe groups DZP, 3ST, 3S and 3S+P with the control L. The results areexpressed as a median and interquartile deviation (Lower Quartile —UpperQuartile).

The statistical and graphical processes were carried out using theSTATVIEW 4.1 and DELTAGRAPH PRO 3.5 software packages.

Results

Overall Stress Score During Test 1

The results of this test are illustrated by FIG. 1.

Legend to FIG. 1

L: Control sterile milk

3ST: Milk fermented by the ferment F042 and then thermized

3S: Milk fermented by the ferment F042

3S+P: Milk fermented by the ferment F042+Passionflower

DZP: Diazepam

Mann-Whitney test: *p<0.05 vs. L, * * * p<0.005 vs. L and (T) trend vs.L (p<0.07).

During test 1, the products 3S and 3S+P significantly reduce the overallstress score compared with the control product L.

The product 3ST is not significantly different from the control productL. Diazepam tends to reduce the overall stress score compared with thecontrol L.

During test 1, apart from diazepam, no product showed a sedative effect.

Overall Stress Score During Test 2

The results of this test are illustrated by FIG. 2.

Legend to FIG. 2

L: Control sterile milk

3ST: Milk fermented by the ferment F042 and then thermized

3S: Milk fermented by the ferment F042

3S+P: Milk fermented by the ferment F042+Passionflower

DZP: Diazepam

Mann-Whitney test: p>0.05 vs. L.

During this test, the products 3S and 3S+P significantly reduce theoverall stress score compared with the control product L.

The effects of the product 3ST and of diazepam are not significantlydifferent from the product L.

Conclusions

During tests 1 and 2, the products 3S and 3S+P significantly reduce theoverall stress score compared with the control product L. The product3ST shows no activity against stress during the two tests compared withthe control product.

Diazepam, administered at the dose of 3 mg/kg p.o., for 6 days beforetest 1, showed only a trend to reduce the overall stress score comparedwith the control. During test 2, it shows no effect which isstatistically different from that of the control. This is a habituationphenomenon specific to products of the benzodiazepine family.

It can be concluded that the products 3S and 3S+P have a significantantistress activity whereas the effect of the product 3ST is notdifferent from that of the control. The phenomenon of tolerance observedwith diazepam is not observed with the products 3S and 3S+P.

No sedation problem was revealed with these two products.

EXAMPLE 3 Anxiolytic Effect of Milks Fermented by Various Lactic AcidBacteria

The products tested are the following:

SC008 1.1: milk fermented by S. thermophilus S242;

SC008 1.2: milk fermented by S. thermophilus S147;

3S: milk fermented by the ferment F042 (S242+S147+S003);

SC008 1.5: filtered whey of milk fermented by the ferment F042;

L: control sterile milk (95° C. 10 min);

SC008 1.8: milk fermented by L. gasseri L012;

SC008 1.10: milk fermented by L. acidophilus L030;

SC008 1.12: milk fermented by S. thermophilus S003.

The tests were carried out according to the protocol described inexample 2 above, on one hundred and fifty eight SPF Wistar/AF males(IFFA-CREDO breeding center, 69—St Germain sur Arbresles, France)weighing 260 to 280 g. After a week's familiarization with thelaboratory conditions, the rats were randomly divided into 13 treatmentgroups. The rats of the various groups were handled in the same mannerand under the same conditions.

Administration of the Products

All the products were orally administered daily for 6 days at the sametimes at the doses of 5 ml/kg using syringes equipped with intragastricforced-feeding probes (HARVARD APPARATUS, 91—Les Ulis, France). On theday for the test, they were administered 6 hours before the buryingtest.

Diazepam (1 mg/kg, p.o., on days 1 and 2; 2 mg/kg p.o., on days 3 and 4and 3 mg/kg, p.o., on days 5 and 6) was suspended in a volume of 5 ml ofaqueous solution of methyl cellulose at 1%. On the day for the test, itwas administered 1 hour before the burying test.

To avoid interference, all the rats of the same cage received the sameproduct.

Variables

In addition to the variables already described in example 2, thefollowing variables of the sedative effect are studied:

number of writhings: the rat adopts the vertical position while restingon its hind legs;

number of passes from one side of the cage to another.

Results

Overall Stress Score

Compared with the product L, the products SC008 1.1, 3S, SC008 1.8,SC008 1.10, SC008 1.12 and diazepam induce significantly lower overallstress scores.

The stress scores induced by the products SC008 1.2 and SC008 1.5 arenot significantly different from the control product. The results areillustrated by FIG. 3.

Legend to FIG. 3:

L: Control sterile milk (95° C. 10 min);

SC008 1.1: milk fermented by S. thermophilus S242;

SC008 1.2: milk fermented by S. thermophilus S147;

3S: milk fermented by the ferment F042 (S242+S147+S003);

SC008 1.5: filtered whey of milk fermented by the ferment F042;

SC008 1.8: milk fermented by L. gasseri L012;

SC008 1.10: milk fermented by L. acidophilus L030;

SC008 1.12: milk fermented by S. thermophilus S003;

DZP: Diazepam.

Mann-Whitney test:

* * * p<0.005 vs.L.

Sedative Effect of the Products 3S and SC008 1.10

The sedative effect of the products 3S and SC008 1.10 compared with thecontrol product L is evaluated on the basis of the locomotive andexploratory activity.

Number of Writhings During the Test

A heterogeneity is observed in the number of writhings obtained with theproducts 3S, SC008 1.10 and L (Kruskal-Wallis test: H_((d.o.f.)2)=6.170; p<0.05). The product 3S causes a significant increase in thenumber of writhings compared with the control product L (Mann-Whitneytest: U=65; p<0.05). The product SC008 1.10 tends to increase the numberof writhings compared with the control product L (Mann-Whitney test:U=73; p<0.07).

Number of Passings From One Side of the Cage to Another

No significant difference is observed in the number of passings from oneside of the cage to another in the case of the products 3S, SC008 1.10and in that of the control product L (Krusdal-Wallis test: H_((d.o.f.)2)0.983: N. S.).

Conclusion

Under the experimental conditions, on the basis of the overall stressscore, SC008 1.1, 3S, SC098 1.8, SC008 1.10, SC008 1.12, and diazepamshow a significant antistress activity compared with the control productL. The products SC008 1.2 and SC008 1.5 exhibit no significantantistress activity compared with the control product L.

On the basis of the locomotive and exploratory activity and comparedwith the control L, no sedative effect was observed with the fermentedmilks showing an antistress activity.

EXAMPLE 4 Determination of the Optimun Duration of Administration of theProducts by the Oral Route

The test products are the following:

3S: Milk fermented by the ferment F042 (S242+S147+S003);

L: Control sterile milk (95° C. 10 min).

The tests were carried out according to the protocol described inexample 2 above, on seventy two SPF Wistar/AF males (Iffa-Credo breedingcenter, 69—St Germain sur Arbresles, France) weighing 260 to 280 g.After a week's familiarization with the laboratory conditions, the ratswere randomly divided into 6 treatment groups. The rats of the variousgroups were handled in the same manner and under the same conditions.

Administration of the Products

The products were orally administered daily for 1, 3 and 6 days at thedoses of 5 ml/kg using syringes equipped with intragastricforced-feeding probes (HARVARD APPARATUS, 91—Les Ulis, France). On theday of the test, they were administered 6 hours before the burying test.

The four rats of each cage all received the same product.

Results

Overall Stress Score

When administered as a single dose or daily for 3 days before the test,the product 3S induces an overall stress score which is statisticallyequivalent to that of the control product L.

When administered daily for 6 days before the test, the product 3Sinduces an overall stress score which is significantly less than thatobtained with the product L.

The results are illustrated by FIG. 4.

Legend to FIG. 4:

L: Control sterile milk (95° C. 10 min)

3S: Milk fermented by the ferment F042 (S242+S147+S003)

Mann-Whitney test:

* * * 0<0.005 vs. L.

Conclusion

Under the experimental conditions described above, a dailyadministration of the product 3S for 6 days is necessary to show asignificant antistress activity compared with the control product L.

EXAMPLE 5 Determination of the Optimun Time for Oral Administration ofthe Products

The test products are the following:

3S: Milk fermented by the ferment F042 (s242+S147+S003)

L: Control sterile milk (95° C. 10 min).

The tests were carried out according to the protocol described inexample 2 above, on one hundred and eight SPF Wistar/AF males(Iffa-Credo breeding center, 69—St Germain sur Arbresles, France)weighing 260 to 280 g. After a week's familiarization with thelaboratory conditions, the rats were randomly divided into 9 treatmentgroups. The rats of the various groups were handled in the same mannerand under the same conditions.

Administration of the Products

Prior to the test, the products were orally administered daily for 6days at the doses of 5 ml/kg using syringes equipped with intragastricforced-feeding probes (Harvard Apparatus, 91—Les Ulis, France). At theend of this period, the burying test is carried out 1, 3 or 6 or 24hours after the last dose.

Diazepam (1 mg/kg, p.o., on days 1 and 2; 2 mg/kg p.o., on days 3 and 4and 3 mg/kg, p.o., on days 5 and 6) is administered in a volume of 5 mlof aqueous solution of methyl cellulose at 1%. The last administrationis carried out 1 hour before the burying test. All the four rats of eachcase received the same product.

Results

Overall Stress Score

When the administration is carried out one hour before the test, theproduct 3S induces an overall stress score equivalent to that obtainedwith the control product L. The overall stress score of diazepam issignificantly lower than that of the control product L.

When the administration is carried out 3 hours before the test, theproduct 3S induces an overall stress score which is not statisticallydifferent from that of the control product L.

When the administration is carried out 6 or 24 hours before the test,the product 3S shows an overall stress score which is significantlylower than that obtained with the control product L.

The results at 1 hour and 6 hours are illustrated by FIGS. 5 and 6.

Legend of FIGS. 5 and 6:

L: Control sterile milk (95° C. 10 min)

3S: Milk fermented by the ferment F042 (S242+S147+S003)

DZP: Diazepam

Mann-Whitney test:

* * p<0.01 vs. L.

Conclusion

Under the experimental conditions described above and on the basis ofthe overall stress score, the antistress activity manifests itselfbetween 6 and 24 hours after the administration of the product 3S.

EXAMPLE 6 Search for the Decapeptide Derived From the Tryptic Hydrolysisof Casein αs1 in a Product Fermented with the 3S Mixture

The presence of the decapeptide described in application EP 95402697 wassought in the product 3S. The analysis was carried out by reversed phaseHPLC on C18 (the separation of the peptides is based on theirhydrophobicity).

The analysis was carried out on a NUCLEOSIL C18-10 μ “precolumn” —100mm×4.6 mm, followed by a NUCLEOSIL C18-3 μ analytical column—150 mm×4.6mm, using the following gradient:

A: TFA 0.1% in H₂O

B: 90% CH₃CN+10% TFA 0.1% in H₂O

The elution is carried out as indicated in table III below.

TABLE III Time A B  0 min 90 10 10 min 60 40 40 min 60 40 45 min 90 1060 min 90 10

The detection is carried out at 215 nm.

Under these conditions, the retention time for the decapeptide depositedon the column is 29 min.

Preparation of the Test Sample:

Product 3S+20% CH₃CN; stirring and then centrifugation 20 min 10 000rpm.

Recovery of the soluble phase and then filtration on GELMAN 0.45 μGHP-GF filter (low protein binding) before injection of 100 μl of thefiltrate onto the HPLC column.

No peak corresponding to the retention time of the desired decapeptidewas found in the filtrate of the product 3S.

What is claimed is:
 1. A method for the production of a nonsedativeanxiolytic which comprises culturing in an appropriate medium at leastone strain of lactic acid bacterium chosen from: the S. thermophilusS242 strain deposited at the CNCM on Feb. 24, 1999 under the numberI-2130; the S. thermophilus S003 strain deposited at the CNCM on Feb.24, 1999 under the number I-2129; the L. gasseri L012 strain depositedat the CNCM on Feb. 24, 1999 under the number I-2131; the L. acidophilusL030 strain deposited at the CNCM on Feb. 24, 1999 under the numberI-2132.
 2. A strain of lactic acid bacterium which can be used for theproduction of a nonsedative anxiolytic, chosen from: the S. thermophilusS242 strain deposited at the CNCM on Feb. 24, 1999 under the numberI-2130; the S. thermophilus S003 strain deposited at the CNCM on Feb.24, 1999 under the number I-2129; the L. gasseri L012 strain depositedat the CNCM on Feb. 24, 1999 under the number I-2131; the L. acidophilusL030 strain deposited at the CNCM on Feb. 24, 1999 under the numberI-2132.
 3. The method as claimed in claim 1, wherein at least oneStreptococcus thermophilus strain chosen from the S242 strain and theS003 strain is combined with the S. thermophilus S147 strain depositedat the CNCM on Dec. 30, 1994 under the number I-1520.
 4. A lacticferment comprising at least one S. thermophilus strain chosen from theS242 strain deposited at the CNCM on Feb. 24, 1999 under the numberI-2130 and the S003 strain deposited at the CNCM on Feb. 24, 1999 underthe number I-2129.
 5. The lactic ferment as claimed in claim 4,comprising, in addition, the S. thermophilus S147 strain deposited atthe CNCM on Dec. 30, 1994 under the number I-1520.
 6. The lactic fermentas claimed in claim 4 which comprises, in addition, at least one strainof lactic acid bacterium chosen from the group consisting of: the L.gasseri L012 strain deposited at the CNCM on Feb. 24, 1999 under thenumber I-2131; the L. acidophilus L030 strain deposited at the CNCM onFeb. 24, 1999 under the number I-2132.
 7. A food product or a dietarysupplement comprising a nonsedative anxiolytic obtained by fermenting amilk or a milk based product with at least one strain of lactic acidbacterium as claimed in claim
 2. 8. The lactic ferment as claimed inclaim 5 which comprises, in addition, at least one strain of lactic acidbacterium chosen from the group consisting of: the L. gasseri L012strain deposited at the CNCM on Feb. 24, 1999 under the number I-2131;the L. acidophilus L030 strain deposited at the CNCM on Feb. 24, 1999under the number I-2132.
 9. The food product or the dietary supplementas claimed in claim 7, which is obtained by fermenting milk or amilk-based medium with a lactic ferment as claimed in claim
 4. 10. Thefood product or the dietary supplement as claimed in claim 7, which isobtained by fermenting milk or a milk-based medium with a lactic fermentas claimed in claim
 5. 11. The food product or the dietary supplement asclaimed in claim 7, which is obtained by fermenting milk or a milk-basedmedium with a lactic ferment as claimed in claim 6.